Methods and compositions for selecting soybean plants resistant to phytophthora root rot

ABSTRACT

The present invention relates to the field of plant breeding and disease resistance. More specifically, the invention includes a method for breeding soybean plants containing quantitative trail loci (QTL) for resistance the  Phytophthora  root rot (PRR) caused by  Phytophthora sojae . The invention further includes the use of molecular markers in the introgression of PRR resistance QTL into soybean plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 60/925,475, filed Apr. 20, 2007, thedisclosure of which application is incorporated herein by reference inits entirety.

INCORPORATION OF THE SEQUENCE LISTING

A sequence listing containing the file named “Sequence Listing.txt”which is 52,115 bytes (measured in Microsoft Windows®) and created onApr. 7, 2008, comprises 116 nucleotide sequences, and is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is in the field of plant breeding and diseaseresistance. More specifically, the invention includes a method ofbreeding plants from the genus Glycine containing quantitative traitloci (QTL) that are associated with disease resistance to the pathogenPhytophthora sojae (Kauffman & Gerdemann). The invention relates to theuse of genetic markers to identify QTL for disease resistance. Theinvention further relates to the use of genetic markers for theintrogression of resistance to Phytophthora sojae into elite germplasmin a breeding program.

BACKGROUND OF THE INVENTION

Phytophthora sojae (Kauffman & Gerdemann) is an oomycete pathogen whichcauses extensive damage to roots and stems of soybean plants (Glycinemax) (Zhang et al., MPMI, 19:1302-1310 (2006)). Symptoms of PhytophthoraRoot Rot (PRR) caused by P. sojae include yellowing and wilting ofleaves and browning of lower stems and branches (Demirbas et al., CropSci. 41:1220-1227 (2001)). PRR results in annual worldwide soybean croplosses of $1 to $2 billion (Zhang et al., MPMI, 19: 1302-1310 (2006)).Soybean PRR resistance or susceptibility depends on a system ofsignaling between pathogen and host. Certain quantitative trait loci(QTL) can confer resistance to PRR. Pathogen avirulence (Avr) and hostresistance (Rps) quantitative trait loci determine the interaction ofdifferent P. sojae races and soybean cultivars (Valer et al., FEMSMicrobiol Lett., 265:60-68 (2006)). Eight loci have been identifiedwhich provide race-specific resistance to PRR, and two of these loci,Rps1 and Rps3, have been identified as having multiple alleles which aredesignated by a letter following the locus number (Ferro et al., CropSci, 46:2427-2436 (2006)). The Rps1 locus includes, for example, Rps1a,Rps1b, Rps1c, Rps1d, and Rps1k, and the Rps3 locus includes, forexample, Rps3a, Rps3b, and Rps3c. Map-based cloning has attempted tocharacterize the Rps1k region (Bhattacharyya, M. K. et al., Theor ApplGenet, 111:75-86 (2005), U.S. Pat. No. 7,256,323). Planting soybeancultivars with race-specific resistance genes has been the primary meansof controlling PRR (Ferro et al., Crop Sci., 46:2427-2436 (2006)). Overfifty P. sojae races have been identified, and Rps loci can provideresistance to more than one P. sojae race. Examples include, but are notlimited to, the following: Rps1k can provide resistance to P. sojaeraces 1 and 4, Rps1c can provide resistance to P. sojae races 1 and 3,and Rps8 can provide resistance to P. sojae races 1, 4, 7, and 25. Plantbreeders are able to use molecular markers as an indirect means toselect plants with alleles resistant to PRR races of concern (Demirbaset al., Crop Sci. 41:1220-1227 (2001)).

Breeding for PRR resistant soybeans can be greatly facilitated by theuse of marker-assisted selection for PRR resistance alleles. Geneticmarkers used in soybean breeding programs to detect, select for, andintrogress PRR resistant plants have included simple sequence repeats(SSRs), restriction fragment length polymorphisms (RFLPs), and singlenucleotide polymorphisms (SNPs). SSR and SNP markers have been providedfor PRR resistance loci on Linkage Groups B1, G, K, and M (U.S. patentapplication Ser. No. 11/199,819 (filed Aug. 8, 2005)). RFLP markers, SSRmarkers, and isozyme markers have been provided for PRR resistance locilocated on Linkage Group A2 (U.S. patent application Ser. No. 10/436,376(filed May 12, 2003)). SSR markers have been provided for PRR resistanceloci located on Linkage Group F (U.S. patent application Ser. No.10/778,018 (filed Feb. 12, 2004)). Linkage groups are described byCregan et al. (Crop Sci. 39:1464-1490 (1999)). To date, a SNP-basedmarker set for Rps1 on Linkage Group N, Rps3 on Linkage Group F, andRps8 on Linkage Group F is lacking.

Of the classes of markers, SNPs have characteristics which make thempreferential to other genetic markers in detecting, selecting for, andintrogressing PRR resistance in a soybean plant. SNPs are preferredbecause technologies are available for automated, high-throughputscreening of SNP markers, which can decrease the time to select for andintrogress PRR resistance in soybean plants. Further, SNP markers areideal because the likelihood that a particular SNP allele is derivedfrom independent origins in the extant population of a particularspecies is very low. As such, SNP markers are useful for tracking andassisting introgression of PRR resistance alleles, particularly in thecase of PRR resistance haplotypes. A need exists for a SNP based markerset to screen for resistance to PRR races with agronomic importance.Rps1, Rps3, and Rps8 provide resistance to PRR races that are asignificant source of damage to soybean crops. The present inventionprovides a SNP-based marker set for Rps1 on Linkage Group N, Rps3 onLinkage Group F, and Rps8 on Linkage Group F.

The present invention provides and includes a method for screening andselecting a soybean plant comprising at least one PRR resistance QTL.The invention includes SNP markers for the detection of, selection for,and introgression of PRR resistance QTL from PRR resistant soybeanplants.

SUMMARY OF THE INVENTION

The present invention includes a method of selecting for andintrogressing an allele into a soybean plant comprising (A) crossing atleast one PRR resistant soybean plant with at least one other soybeanplant in order to form a population, (B) screening said population withat least one nucleic acid marker selected from the group comprising SEQID NO: 1 to SEQ ID NO: 16 and SEQ ID NO: 81 to SEQ ID NO: 84, (C)selecting from said population one or more soybean plants comprising atleast one genotype corresponding to a PRR resistant soybean plant.

The present invention further comprises an elite soybean plant producedby such method.

The present invention includes a method of introgressing an allele intoa soybean plant comprising: (A) crossing at least one PRR resistantsoybean plant with at least one other soybean plant in order to form apopulation, (B) screening said population with at least one nucleic acidmarker, (C) selecting from said population one or more soybean plantscomprising a haplotype associated with PRR resistance, wherein said PRRresistance haplotype is selected from the group consisting of 1, 2, or 3PRR resistant loci where one or more haplotypes at one or more loci areselected from the group of Rps1, Rps3, and Rps8, and the one or morehaplotypes are selected based on the haplotype of the PRR resistantsoybean plants.

The present invention further comprises an elite soybean plant producedby said method.

The present invention includes a substantially purified nucleic acidmolecule comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 116.

Further, the present invention provides assays for detection of the PRRresistance QTLs, Rps1, Rps3, and Rps8.

BRIEF DESCRIPTION OF THE NUCLEIC ACID SEQUENCES

SEQ ID NO: 1 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 2 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 3 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 4 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 5 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 6 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 7 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 8 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 9 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 10 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 11 is a genomic sequence derived from Glycine max associatedwith the PRR resistance loci Rps3 and Rps8.

SEQ ID NO: 12 is a genomic sequence derived from Glycine max associatedwith the PRR resistance loci Rps3 and Rps8.

SEQ ID NO: 13 is a genomic sequence derived from Glycine max associatedwith the PRR resistance loci Rps3 and Rps8.

SEQ ID NO: 14 is a genomic sequence derived from Glycine max associatedwith the PRR resistance loci Rps3 and Rps8.

SEQ ID NO: 15 is a genomic sequence derived from Glycine max associatedwith the PRR resistance loci Rps3 and Rps8.

SEQ ID NO: 16 is a genomic sequence derived from Glycine max associatedwith the PRR resistance loci Rps3 and Rps8.

SEQ ID NO: 17 is a forward PCR primer for amplifying SEQ ID NO: 1.

SEQ ID NO: 18 is a reverse PCR primer for amplifying SEQ ID NO: 1.

SEQ ID NO: 19 is a forward PCR primer for amplifying SEQ ID NO: 2.

SEQ ID NO: 20 is a reverse PCR primer for amplifying SEQ ID NO: 2.

SEQ ID NO: 21 is a forward PCR primer for amplifying SEQ ID NO: 3.

SEQ ID NO: 22 is a reverse PCR primer for amplifying SEQ ID NO: 3.

SEQ ID NO: 23 is a forward PCR primer for amplifying SEQ ID NO: 4.

SEQ ID NO: 24 is a reverse PCR primer for amplifying SEQ ID NO: 4.

SEQ ID NO: 25 is a forward PCR primer for amplifying SEQ ID NO: 5.

SEQ ID NO: 26 is a reverse PCR primer for amplifying SEQ ID NO: 5.

SEQ ID NO: 27 is a forward PCR primer for amplifying SEQ ID NO: 6.

SEQ ID NO: 28 is a reverse PCR primer for amplifying SEQ ID NO: 6.

SEQ ID NO: 29 is a forward PCR primer for amplifying SEQ ID NO: 7.

SEQ ID NO: 30 is a reverse PCR primer for amplifying SEQ ID NO: 7.

SEQ ID NO: 31 is a forward PCR primer for amplifying SEQ ID NO: 8.

SEQ ID NO: 32 is a reverse PCR primer for amplifying SEQ ID NO: 8.

SEQ ID NO: 33 is a forward PCR primer for amplifying SEQ ID NO: 9.

SEQ ID NO: 34 is a reverse PCR primer for amplifying SEQ ID NO: 9.

SEQ ID NO: 35 is a forward PCR primer for amplifying SEQ ID NO: 10.

SEQ ID NO: 36 is a reverse PCR primer for amplifying SEQ ID NO: 10.

SEQ ID NO: 37 is a forward PCR primer for amplifying SEQ ID NO: 11.

SEQ ID NO: 38 is a reverse PCR primer for amplifying SEQ ID NO: 11.

SEQ ID NO: 39 is a forward PCR primer for amplifying SEQ ID NO: 12.

SEQ ID NO: 40 is a reverse PCR primer for amplifying SEQ ID NO: 12.

SEQ ID NO: 41 is a forward PCR primer for amplifying SEQ ID NO: 13.

SEQ ID NO: 42 is a reverse PCR primer for amplifying SEQ ID NO: 13.

SEQ ID NO: 43 is a forward PCR primer for amplifying SEQ ID NO: 14.

SEQ ID NO: 44 is a reverse PCR primer for amplifying SEQ ID NO: 14.

SEQ ID NO: 45 is a forward PCR primer for amplifying SEQ ID NO: 15.

SEQ ID NO: 46 is a reverse PCR primer for amplifying SEQ ID NO: 15.

SEQ ID NO: 47 is a forward PCR primer for amplifying SEQ ID NO: 16.

SEQ ID NO: 48 is a reverse PCR primer for amplifying SEQ ID NO: 16.

SEQ ID NO: 49 is a probe for detecting the PRR resistance locus of SEQID NO: 1.

SEQ ID NO: 50 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 1.

SEQ ID NO: 51 is a probe for detecting the PRR resistance locus of SEQID NO: 2.

SEQ ID NO: 52 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 2.

SEQ ID NO: 53 is a probe for detecting the PRR resistance locus of SEQID NO: 3.

SEQ ID NO: 54 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 3.

SEQ ID NO: 55 is a probe for detecting the PRR resistance locus of SEQID NO: 4.

SEQ ID NO: 56 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 4.

SEQ ID NO: 57 is a probe for detecting the PRR resistance locus of SEQID NO: 5.

SEQ ID NO: 58 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 5.

SEQ ID NO: 59 is a probe for detecting the PRR resistance locus of SEQID NO: 6.

SEQ ID NO: 60 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 6.

SEQ ID NO: 61 is a probe for detecting the PRR resistance locus of SEQID NO: 7.

SEQ ID NO: 62 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 7.

SEQ ID NO: 63 is a probe for detecting the PRR resistance locus of SEQID NO: 8.

SEQ ID NO: 64 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 8.

SEQ ID NO: 65 is a probe for detecting the PRR resistance locus of SEQID NO: 9.

SEQ ID NO: 66 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 9.

SEQ ID NO: 67 is a probe for detecting the PRR resistance locus of SEQID NO: 10.

SEQ ID NO: 68 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 10.

SEQ ID NO: 69 is a probe for detecting the PRR resistance loci of SEQ IDNO: 11.

SEQ ID NO: 70 is a second probe for detecting the PRR resistance loci ofSEQ ID NO: 11.

SEQ ID NO: 71 is a probe for detecting the PRR resistance loci of SEQ IDNO: 12.

SEQ ID NO: 72 is a second probe for detecting the PRR resistance loci ofSEQ ID NO: 12.

SEQ ID NO: 73 is a probe for detecting the PRR resistance loci of SEQ IDNO: 13.

SEQ ID NO: 74 is a second probe for detecting the PRR resistance loci ofSEQ ID NO: 13.

SEQ ID NO: 75 is a probe for detecting the PRR resistance loci of SEQ IDNO: 14.

SEQ ID NO: 76 is a second probe for detecting the PRR resistance loci ofSEQ ID NO: 14.

SEQ ID NO: 77 is a probe for detecting the PRR resistance loci of SEQ IDNO: 15.

SEQ ID NO: 78 is a second probe for detecting the PRR resistance loci ofSEQ ID NO: 15.

SEQ ID NO: 79 is a probe for detecting the PRR resistance loci of SEQ IDNO: 16.

SEQ ID NO: 80 is a second probe for detecting the PRR resistance loci ofSEQ ID NO: 16.

SEQ ID NO: 81 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 82 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 83 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 84 is a genomic sequence derived from Glycine max associatedwith the PRR resistance locus Rps1.

SEQ ID NO: 85 is a forward PCR primer for amplifying SEQ ID NO: 81.

SEQ ID NO: 86 is a reverse PCR primer for amplifying SEQ ID NO: 81.

SEQ ID NO: 87 is a forward PCR primer for amplifying SEQ ID NO: 82.

SEQ ID NO: 88 is a reverse PCR primer for amplifying SEQ ID NO: 82.

SEQ ID NO: 89 is a forward PCR primer for amplifying SEQ ID NO: 83.

SEQ ID NO: 90 is a reverse PCR primer for amplifying SEQ ID NO: 83.

SEQ ID NO: 91 is a forward PCR primer for amplifying SEQ ID NO: 84.

SEQ ID NO: 92 is a reverse PCR primer for amplifying SEQ ID NO: 84.

SEQ ID NO: 93 is a probe for detecting the PRR resistance locus of SEQID NO: 81.

SEQ ID NO: 94 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 81.

SEQ ID NO: 95 is a probe for detecting the PRR resistance locus of SEQID NO: 82.

SEQ ID NO: 96 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 82.

SEQ ID NO: 97 is a probe for detecting the PRR resistance locus of SEQID NO: 83.

SEQ ID NO: 98 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 83.

SEQ ID NO: 99 is a probe for detecting the PRR resistance locus of SEQID NO: 84.

SEQ ID NO: 100 is a second probe for detecting the PRR resistance locusof SEQ ID NO: 84.

SEQ ID NO: 101 is a third probe for detecting the PRR resistance locusof SEQ ID NO: 8.

SEQ ID NO: 102 is a fourth probe for detecting the PRR resistance locusof SEQ ID NO: 8.

SEQ ID NO: 103 is a third probe for detecting the PRR resistance locusof SEQ ID NO: 10.

SEQ ID NO: 104 is a fourth probe for detecting the PRR resistance locusof SEQ ID NO: 10.

SEQ ID NO: 105 is a third probe for detecting the PRR resistance loci ofSEQ ID NO: 14.

SEQ ID NO: 106 is a fourth probe for detecting the PRR resistance lociof SEQ ID NO: 14.

SEQ ID NO: 107 is a third probe for detecting the PRR resistance loci ofSEQ ID NO: 15.

SEQ ID NO: 108 is a fourth probe for detecting the PRR resistance lociof SEQ ID NO: 15.

SEQ ID NO: 109 is a fifth probe for detecting the PRR resistance locusof SEQ ID NO: 8.

SEQ ID NO: 110 is a sixth probe for detecting the PRR resistance locusof SEQ ID NO: 8.

SEQ ID NO: 111 is a fifth probe for detecting the PRR resistance locusof SEQ ID NO: 10.

SEQ ID NO: 112 is a sixth probe for detecting the PRR resistance locusof SEQ ID NO: 10.

SEQ ID NO: 113 is a fifth probe for detecting the PRR resistance loci ofSEQ ID NO: 14.

SEQ ID NO: 114 is a sixth probe for detecting the PRR resistance loci ofSEQ ID NO: 14.

SEQ ID NO: 115 is a fifth probe for detecting the PRR resistance locusof SEQ ID NO: 15.

SEQ ID NO: 116 is a sixth probe for detecting the PRR resistance locusof SEQ ID NO: 15.

DETAILED DESCRIPTION OF THE INVENTION

The definitions and methods provided define the present invention andguide those of ordinary skill in the art in the practice of the presentinvention. Unless otherwise noted, terms are to be understood accordingto conventional usage by those of ordinary skill in the relevant art.Definitions of common terms in molecular biology may also be found inAlberts et al., Molecular Biology of The Cell, 3^(rd) Edition, GarlandPublishing, Inc.: New York, 1994; Rieger et al., Glossary of Genetics:Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991;and Lewin, Genes V, Oxford University Press: New York, 1994. Thenomenclature for DNA bases as set forth at 37 CFR § 1.822 is used.

An “allele” refers to an alternative sequence at a particular locus; thelength of an allele can be as small as 1 nucleotide base, but istypically larger.

A “locus” is a position on a genomic sequence that is usually found by apoint of reference; e.g., a short DNA sequence that is a gene, or partof a gene or intergenic region. The loci of this invention comprise oneor more polymorphisms in a population; i.e., alternative alleles presentin some individuals.

As used herein, “polymorphism” means the presence of one or morevariations of a nucleic acid sequence at one or more loci in apopulation of one or more individuals. The variation may comprise but isnot limited to one or more base changes, the insertion of one or morenucleotides or the deletion of one or more nucleotides. A polymorphismmay arise from random processes in nucleic acid replication, throughmutagenesis, as a result of mobile genomic elements, from copy numbervariation and during the process of meiosis, such as unequal crossingover, genome duplication and chromosome breaks and fusions. Thevariation can be commonly found or may exist at low frequency within apopulation, the former having greater utility in general plant breedingand the latter may be associated with rare but important phenotypicvariation. Useful polymorphisms may include single nucleotidepolymorphisms (SNPs), insertions or deletions in DNA sequence (Indels),simple sequence repeats of DNA sequence (SSRs) a restriction fragmentlength polymorphism, and a tag SNP. A genetic marker, a gene, aDNA-derived sequence, a haplotype, a RNA-derived sequence, a promoter, a5′ untranslated region of a gene, a 3′ untranslated region of a gene,microRNA, siRNA, a QTL, a satellite marker, a transgene, mRNA, ds mRNA,a transcriptional profile, and a methylation pattern may comprisepolymorphisms.

As used herein, “marker” means a polymorphic nucleic acid sequence ornucleic acid feature. A marker may be represented by one or moreparticular variant sequences, or by a consensus sequence. In anothersense, a “marker” is an isolated variant or consensus of such asequence. In a broader aspect, a “marker” can be a detectablecharacteristic that can be used to discriminate between heritabledifferences between organisms. Examples of such characteristics mayinclude genetic markers, protein composition, protein levels, oilcomposition, oil levels, carbohydrate composition, carbohydrate levels,fatty acid composition, fatty acid levels, amino acid composition, aminoacid levels, biopolymers, pharmaceuticals, starch composition, starchlevels, fermentable starch, fermentation yield, fermentation efficiency,energy yield, secondary compounds, metabolites, morphologicalcharacteristics, and agronomic characteristics.

As used herein, “marker assay” means a method for detecting apolymorphism at a particular locus using a particular method, e.g.measurement of at least one phenotype (such as seed color, flower color,or other visually detectable trait), restriction fragment lengthpolymorphism (RFLP), single base extension, electrophoresis, sequencealignment, allelic specific oligonucleotide hybridization (ASO), randomamplified polymorphic DNA (RAPD), microarray-based technologies, andnucleic acid sequencing technologies, etc.

As used herein, “typing” refers to any method whereby the specificallelic form of a given soybean genomic polymorphism is determined. Forexample, a single nucleotide polymorphism (SNP) is typed by determiningwhich nucleotide is present (i.e. an A, G, T, or C). Insertion/deletions(Indels) are determined by determining if the Indel is present. Indelscan be typed by a variety of assays including, but not limited to,marker assays.

As used herein, the term “adjacent”, when used to describe a nucleicacid molecule that hybridizes to DNA containing a polymorphism, refersto a nucleic acid that hybridizes to DNA sequences that directly abutthe polymorphic nucleotide base position. For example, a nucleic acidmolecule that can be used in a single base extension assay is “adjacent”to the polymorphism.

As used herein, “interrogation position” refers to a physical positionon a solid support that can be queried to obtain genotyping data for oneor more predetermined genomic polymorphisms.

As used herein, “consensus sequence” refers to a constructed DNAsequence which identifies SNP and Indel polymorphisms in alleles at alocus. Consensus sequence can be based on either strand of DNA at thelocus and states the nucleotide base of either one of each SNP in thelocus and the nucleotide bases of all Indels in the locus. Thus,although a consensus sequence may not be a copy of an actual DNAsequence, a consensus sequence is useful for precisely designing primersand probes for actual polymorphisms in the locus.

As used herein, the term “single nucleotide polymorphism,” also referredto by the abbreviation “SNP,” means a polymorphism at a single sitewherein said polymorphism constitutes a single base pair change, aninsertion of one or more base pairs, or a deletion of one or more basepairs.

As used herein, the term “haplotype” means a chromosomal region within ahaplotype window defined by at least one polymorphic molecular marker.The unique marker fingerprint combinations in each haplotype windowdefine individual haplotypes for that window. Further, changes in ahaplotype, brought about by recombination for example, may result in themodification of a haplotype so that it comprises only a portion of theoriginal (parental) haplotype operably linked to the trait, for example,via physical linkage to a gene, QTL, or transgene. Any such change in ahaplotype would be included in our definition of what constitutes ahaplotype so long as the functional integrity of that genomic region isunchanged or improved.

As used herein, the term “haplotype window” means a chromosomal regionthat is established by statistical analyses known to those of skill inthe art and is in linkage disequilibrium. Thus, identity by statebetween two inbred individuals (or two gametes) at one or more molecularmarker loci located within this region is taken as evidence ofidentity-by-descent of the entire region. Each haplotype window includesat least one polymorphic molecular marker. Haplotype windows can bemapped along each chromosome in the genome. Haplotype windows are notfixed per se and, given the ever-increasing density of molecularmarkers, this invention anticipates the number and size of haplotypewindows to evolve, with the number of windows increasing and theirrespective sizes decreasing, thus resulting in an ever-increasing degreeconfidence in ascertaining identity by descent based on the identity bystate at the marker loci.

As used herein, “genotype” means the genetic component of the phenotypeand it can be indirectly characterized using markers or directlycharacterized by nucleic acid sequencing. Suitable markers include aphenotypic character, a metabolic profile, a genetic marker, or someother type of marker. A genotype may constitute an allele for at leastone genetic marker locus or a haplotype for at least one haplotypewindow. In some embodiments, a genotype may represent a single locus andin others it may represent a genome-wide set of loci. In anotherembodiment, the genotype can reflect the sequence of a portion of achromosome, an entire chromosome, a portion of the genome, and theentire genome.

As used herein, “phenotype” means the detectable characteristics of acell or organism which can be influenced by gene expression.

As used herein, “linkage” refers to relative frequency at which types ofgametes are produced in a cross. For example, if locus A has genes “A”or “a” and locus B has genes “B” or “b” and a cross between parent Iwith AABB and parent B with aabb will produce four possible gameteswhere the genes are segregated into AB, Ab, aB and ab. The nullexpectation is that there will be independent equal segregation intoeach of the four possible genotypes, i.e. with no linkage 1/4 of thegametes will of each genotype. Segregation of gametes into a genotypesdiffering from 1/4 are attributed to linkage.

As used herein, “linkage disequilibrium” is defined in the context ofthe relative frequency of gamete types in a population of manyindividuals in a single generation. If the frequency of allele A is p, ais p′, B is q and b is q′, then the expected frequency (with no linkagedisequilibrium) of genotype AB is pq, Ab is pq′, aB is p′q and ab isp′q′. Any deviation from the expected frequency is called linkagedisequilibrium. Two loci are said to be “genetically linked” when theyare in linkage disequilibrium.

As used herein, “quantitative trait locus (QTL)” means a locus thatcontrols to some degree numerically representable traits that areusually continuously distributed.

As used herein, “resistance allele” means the isolated nucleic acidsequence that includes the polymorphic allele associated with resistanceto PRR.

As used herein, the term “soybean” means Glycine max and includes allplant varieties that can be bred with soybean, including wild soybeanspecies.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, the term “elite line” means any line that has resultedfrom breeding and selection for superior agronomic performance. An eliteplant is any plant from an elite line. Non-limiting examples of elitesoybean varieties that are commercially available to farmers or soybeanbreeders include AG00802, A0868, AG0902, A1923, AG2403, A2824, A3704,A4324, A5404, AG5903 and AG6202 (Asgrow Seeds, Des Moines, Iowa, USA);BPR0144RR, BPR 4077NRR and BPR 4390NRR (Bio Plant Research, Camp Point,Ill., USA); DKB17-51 and DKB37-51 (DeKalb Genetics, DeKalb, Ill., USA);and DP 4546 RR, and DP 7870 RR (Delta & Pine Land Company, Lubbock,Tex., USA); JG 03R501, JG 32R606C ADD and JG 55R503C (JGL Inc.,Greencastle, Ind., USA); NKS13-K2 (NK Division of Syngenta Seeds, GoldenValley, Minn., USA); 90M01, 91M30, 92M33, 93M11, 94M30, 95M30 and 97B52(Pioneer Hi-Bred International, Johnston, Iowa, USA); SG4771NRR andSG5161NRR/STS (Soygenetics, LLC, Lafayette, Ind., USA); S00-K5, S11-L2,S28-Y2, S43-B1, S53-A1, S76-L9 and S78-G6 (Syngenta Seeds, Henderson,Ky., USA). An elite plant is a representative plant from an elitevariety.

The present invention provides SNP genetic markers useful for screeningand selecting for PRR resistance at the Rps1 locus located on LinkageGroup N (LG N) (Cregan, et al. Crop Sci. 39:1464-1490 (1999)). Thepresent invention also provides SNP DNA markers useful for screening andselecting for PRR resistance at the Rps3 locus located on Linkage GroupF (LG F) and the Rps8 locus located on Linkage Group F (LG F) (Cregan,et al. Crop Sci. 39:1464-1490 (1999)). The SNP markers are useful formonitoring the selection for and introgression of the PRR resistanceloci from PRR resistant sources. As used herein, the Rps1 locusincludes, for example, Rps1a, Rps1b, Rps1c, Rps1d, and Rps1k. Further,as used herein, the Rps3 locus includes, for example, Rps3a, Rps3b, andRps3c.

The present invention also includes a method of selecting orintrogressing a PRR resistant allele into a soybean plant comprising:(A) crossing at least one PRR resistant soybean plant with at least oneother soybean plant in order to form a population; (B) screening thepopulation with one or more nucleic acid markers to determine if one ormore soybean plants from the population contains the allele of the PRRresistance source.

SNP markers used to monitor the selection or introgression of the PRRresistance locus 1 (Rps1) include those selected from the groupconsisting of NS0099413, NS0102174, NS0118166, NS0102920, NS0114258,NS0118976, NS0119981, NS0119335, NS0201536, NS0138011, NS0202603,NS0203225, NS0129030, and NS0127084. Sources of Rps1 include bothaccession germplasm, such as plant introductions, and elite germplasm.Sources include, but are not limited to, Williams 82, L75-3735, andelite varieties with demonstrated PRR resistance. PRR resistance SNPmarker DNA sequences (SEQ ID NO: 1 through 10 and SEQ ID NO: 81 through84) can be amplified using the primers indicated as SEQ ID NO: 17through 36 and SEQ ID NO: 85 through 92 and detected with probesindicated as SEQ ID NO: 49 through 68 and SEQ ID NO: 93 through 100,wherein the corresponding primer and probe sets provide assays for thedetection of PRR resistance or susceptibility in Glycine max.Determination of resistance or susceptibility of a plant to a particularpathogen is obvious to anyone skilled in the art.

In the present invention, a PRR resistance locus 3 (Rps3) is located onLinkage Group F (Cregan, et al. Crop Sci. 39:1464-1490 (1999)). SNPmarkers used to monitor the introgression of Rps3 can be selected fromthe group consisting of NS0114683, NS0101324, NS0102483, NS0119333,NS0102262, and NS0116265. Sources of Rps3 include both accession andelite germplasm. Sources of Rps3 include, but are not limited to,L83-570, L89-1541, L92-7857, Ivory, and elite varieties withdemonstrated PRR resistance. In the present invention, a PRR resistancelocus 8 (Rps8) is located on Linkage Group F (Cregan, et al. Crop Sci.39:1464-1490 (1999)). SNP markers used to monitor the introgression ofPRR resistance locus 8 can be selected from the group consisting ofNS0114683, NS0101324, NS0102483, NS0119333, NS0102262, and NS0116265.Sources of Rps8 include accession germplasm. Sources of Rps8 include,but are not limited to, PI399703 and other varieties with known PRRresistance.

In one aspect, the present invention provides methods and compositionsfor screening soybean plants for resistance or susceptibility to PRR,caused by the species Phytophthora sojae. In another aspect, the presentinvention provides methods and compositions for selecting PRR resistantplants. In a preferred aspect, the present invention provides methodsand compositions for selecting for and introgressing PRR resistance intosoybean plants. The PRR resistance alleles of the present invention maybe introduced into an elite Glycine max line.

As used herein, PRR refers to any PRR race, variant or isolate. Asoybean plant of the present invention can be resistant to one or moreoomycete capable of causing or inducing PRR. In one aspect, the presentinvention provides plants resistant to and methods and compositions forscreening soybean plants for resistance or susceptibility toPhytophthora sojae races 1 through 55. In another aspect, the presentinvention provides plants resistant to and methods and compositions forscreening soybean plants for resistance or susceptibility toPhytophthora sojae race 1. In additional aspect, the present inventionprovides plants resistant to and methods and compositions for screeningsoybean plants for resistance or susceptibility to Phytophthora sojaerace 3. In a further aspect, the present invention provides plantsresistant to and methods and compositions for screening soybean plantsfor resistance or susceptibility to Phytophthora sojae race 4. Theinvention further provides plants resistant to and methods andcompositions for screening soybean plants for resistance orsusceptibility to Phytophthora sojae race 7. The invention furtherprovides plants resistant to and methods and compositions for screeningsoybean plants for resistance or susceptibility to Phytophthora sojaerace 17. The invention further provides plants resistant to and methodsand compositions for screening soybean plants for resistance orsusceptibility to Phytophthora sojae race 25.

The PRR resistance alleles of the present invention may also beintroduced into an elite Glycine max transgenic plant that contains oneor more genes for herbicide tolerance, increased yield, insect control,fungal disease resistance, virus resistance, nematode resistance,bacterial disease resistance, mycoplasma disease resistance, modifiedoils production, high oil production, high protein production,germination and seedling growth control, enhanced animal and humannutrition, low raffinose, environmental stress resistance, increaseddigestibility, industrial enzymes, pharmaceutical proteins, peptides andsmall molecules, improved processing traits, improved flavor, nitrogenfixation, hybrid seed production, reduced allergenicity, biopolymers,and biofuels among others. These agronomic traits can be provided by themethods of plant biotechnology as transgenes in Glycine max.

A disease resistance allele or alleles can be introduced from any plantthat contains that allele (donor) to any recipient soybean plant. In oneaspect, the recipient soybean plant can contain additional PRRresistance loci. In another aspect, the recipient soybean plant cancontain a transgene. In another aspect, while maintaining the introducedPRR resistance allele, the genetic contribution of the plant providingthe disease resistance allele can be reduced by back-crossing or othersuitable approaches. In one aspect, the nuclear genetic material derivedfrom the donor material in the soybean plant can be less than or about50%, less than or about 25%, less than or about 13%, less than or about5%, 3%, 2% or 1%, but that genetic material contains the PRR resistancelocus or loci of interest.

It is further understood that a soybean plant of the present inventionmay exhibit the characteristics of any relative maturity group. In anaspect, the maturity group is selected from the group consisting of 000,00, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

The present invention also provides for parts of the plants of thepresent invention. Plant parts, without limitation, include seed,endosperm, ovule and pollen. In a particularly preferred aspect of thepresent invention, the plant part is a seed.

Plants or parts thereof of the present invention may be grown in cultureand regenerated. Methods for the regeneration of Glycine max plants fromvarious tissue types and methods for the tissue culture of Glycine maxare known in the art (See, for example, Widholm et al., In VitroSelection and Culture-induced Variation in Soybean, In Soybean:Genetics, Molecular Biology and Biotechnology, Eds. Verma and Shoemaker,CAB International, Wallingford, Oxon, England (1996). Regenerationtechniques for plants such as Glycine max can use as the startingmaterial a variety of tissue or cell types. With Glycine max inparticular, regeneration processes have been developed that begin withcertain differentiated tissue types such as meristems, Cartha et al.,Can. J. Bot. 59:1671-1679 (1981), hypocotyl sections, Cameya et al.,Plant Science Letters 21: 289-294 (1981), and stem node segments, Sakaet al., Plant Science Letters, 19: 193-201 (1980); Cheng et al., PlantScience Letters, 19: 91-99 (1980). Regeneration of whole sexually matureGlycine max plants from somatic embryos generated from explants ofimmature Glycine max embryos has been reported (Ranch et al., In VitroCellular & Developmental Biology 21: 653-658 (1985). Regeneration ofmature Glycine max plants from tissue culture by organogenesis andembryogenesis has also been reported (Barwale et al., Planta 167:473-481 (1986); Wright et al., Plant Cell Reports 5: 150-154 (1986).

Plants containing one or more PRR resistance loci described can be donorplants. Soybean plants containing resistance loci can be, for example,screened for by using a nucleic acid molecule capable of detecting amarker polymorphism associated or genetically linked with each of theresistance alleles.

As used herein, an allele of a disease resistance locus can encompassmore than one gene or other genetic factor where each individual gene orgenetic component is also capable of exhibiting allelic variation andwhere each gene or genetic factor is also capable of eliciting aphenotypic effect on the quantitative trait in question. In an aspect ofthe present invention the resistance allele comprises one or more genesor other genetic factors that are also capable of exhibiting allelicvariation. The use of the term “a resistance allele” does not exclude agenomic region that comprises more than one gene or other geneticfactor. Specifically, a “disease resistance allele” in the present inthe invention can denote a haplotype allele within a haplotype window orgenomic region wherein a phenotype associated with said haplotype allelecan be disease resistance. A haplotype window is a contiguous genomicregion that can be defined, and tracked, with a set of one or morepolymorphic markers wherein the polymorphisms indicate identity bydescent. A haplotype within that window can be defined by the uniquefingerprint of alleles at each marker. When all the alleles present at agiven locus on a chromosome are the same, that plant is homozygous atthat locus. If the alleles present at a given locus on a chromosomediffer, that plant is heterozygous at that locus. Plants of the presentinvention may be homozygous or heterozygous at any particular PRRresistance locus or for a particular polymorphic marker.

The present invention includes isolated nucleic acid molecules. Suchmolecules include those nucleic acid molecules capable of detecting apolymorphism genetically or physically linked to a PRR resistance locus.Additional markers can be obtained that are genetically linked to Rps1,Rps3, and Rps8, by available techniques. In one aspect, the nucleic acidmolecule is capable of detecting the presence or absence of a markerlocated less than 30, 25, 20, 10, 5, 2, or 1 centimorgans from a PRRresistance locus. In another aspect, the nucleic acid molecule iscapable of detecting a marker in a locus selected from the group Rps1,Rps3, and Rps8. In a further aspect, a nucleic acid molecule is selectedfrom the group consisting of SEQ ID NO: 1 to SEQ ID NO: 116, fragmentsthereof, complements thereof, and nucleic acid molecules capable ofspecifically hybridizing to one or more of these nucleic acid molecules.

In a preferred aspect, a nucleic acid molecule of the present inventionincludes those that will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NO: 1 through SEQ ID NO: 116or complements thereof or fragments of either under moderately stringentconditions, for example at about 2.0×SSC and about 65° C. In aparticularly preferred aspect, a nucleic acid of the present inventionwill specifically hybridize to one or more of the nucleic acid moleculesset forth in SEQ ID NO: 1 through SEQ ID NO: 116 or complements orfragments of either under high stringency conditions. In one aspect ofthe present invention, a preferred marker nucleic acid molecule of thepresent invention has the nucleic acid sequence set forth in SEQ ID NO:1 through SEQ ID NO: 116 or complements thereof or fragments of either.In another aspect of the present invention, a preferred marker nucleicacid molecule of the present invention shares between 80% and 100% or90% and 100% sequence identity with the nucleic acid sequence set forthin SEQ ID NO: 1 through SEQ ID NO: 116 or complement thereof orfragments of either. In a further aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 95% and 100% sequence identity with the sequence set forth inSEQ ID NO: 1 through SEQ ID NO: 116 or complement thereof or fragmentsof either. In a more preferred aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 98% and 100% sequence identity with the nucleic acid sequenceset forth in SEQ ID NO: 1 through SEQ ID NO: 116 or complement thereofor fragments of either.

Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is the “complement” of another nucleic acid molecule if theyexhibit complete complementarity. As used herein, molecules exhibit“complete complementarity” when every nucleotide of one of the moleculesis complementary to a nucleotide of the other. Two molecules are“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., In: Molecular Cloning, ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and by Haymes et al., In: Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. In order for a nucleicacid molecule to serve as a primer or probe it need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The nucleic-acid probes and primers of thepresent invention can hybridize under stringent conditions to a targetDNA sequence. The term “stringent hybridization conditions” is definedas conditions under which a probe or primer hybridizes specifically witha target sequence(s) and not with non-target sequences, as can bedetermined empirically. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at9.47-9.52, 9.56-9.58; Kanehisa 1984 Nucl. Acids Res. 12:203-213; andWetmur et al. 1968 J. Mol. Biol. 31:349-370. Appropriate stringencyconditions that promote DNA hybridization are, for example, 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C., are known to those skilled in the art or can be foundin Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.,1989, 6.3.1-6.3.6. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged.

For example, hybridization using DNA or RNA probes or primers can beperformed at 65° C. in 6×SSC, 0.5% SDS, 5×Denhardt's, 100 μg/mLnonspecific DNA (e.g., sonicated salmon sperm DNA) with washing at0.5×SSC, 0.5% SDS at 65° C., for high stringency.

It is contemplated that lower stringency hybridization conditions suchas lower hybridization and/or washing temperatures can be used toidentify related sequences having a lower degree of sequence similarityif specificity of binding of the probe or primer to target sequence(s)is preserved. Accordingly, the nucleotide sequences of the presentinvention can be used for their ability to selectively form duplexmolecules with complementary stretches of DNA, RNA, or cDNA fragments.

A fragment of a nucleic acid molecule can be any sized fragment andillustrative fragments include fragments of nucleic acid sequences setforth in SEQ ID NO: 1 to SEQ ID NO: 116 and complements thereof. In oneaspect, a fragment can be between 15 and 25, 15 and 30, 15 and 40, 15and 50, 15 and 100, 20 and 25, 20 and 30, 20 and 40, 20 and 50, 20 and100, 25 and 30, 25 and 40, 25 and 50, 25 and 100, 30 and 40, 30 and 50,and 30 and 100. In another aspect, the fragment can be greater than 10,15, 20, 25, 30, 35, 40, 50, 100, or 250 nucleotides.

Additional genetic markers can be used to select plants with an alleleof a QTL associated with disease resistance of soybean of the presentinvention. Examples of public marker databases include, for example,Soybase, an Agricultural Research Service, United States Department ofAgriculture.

Genetic markers of the present invention include “dominant” or“codominant” markers. “Codominant markers” reveal the presence of two ormore alleles (two per diploid individual). “Dominant markers” reveal thepresence of only a single allele. The presence of the dominant markerphenotype (e.g., a band of DNA) is an indication that one allele ispresent in either the homozygous or heterozygous condition. The absenceof the dominant marker phenotype (e.g., absence of a DNA band) is merelyevidence that “some other” undefined allele is present. In the case ofpopulations where individuals are predominantly homozygous and loci arepredominantly dimorphic, dominant and codominant markers can be equallyvaluable. As populations become more heterozygous and multiallelic,codominant markers often become more informative of the genotype thandominant markers.

In another embodiment, markers, such as single sequence repeat markers(SSR), AFLP markers, RFLP markers, RAPD markers, phenotypic markers,isozyme markers, single nucleotide polymorphisms (SNPs), insertions ordeletions (Indels), single feature polymorphisms (SFPs, for example, asdescribed in Borevitz et al. 2003 Gen. Res. 13:513-523), microarraytranscription profiles, DNA-derived sequences, and RNA-derived sequencesthat are genetically linked to or correlated with alleles of a QTL ofthe present invention can be utilized.

In one embodiment, nucleic acid-based analyses for the presence orabsence of the genetic polymorphism can be used for the selection ofseeds in a breeding population. A wide variety of genetic markers forthe analysis of genetic polymorphisms are available and known to thoseof skill in the art. The analysis may be used to select for genes, QTL,alleles, or genomic regions (haplotypes) that comprise or are linked toa genetic marker.

Herein, nucleic acid analysis methods are known in the art and include,but are not limited to, PCR-based detection methods (for example, TaqManassays), microarray methods, and nucleic acid sequencing methods. In oneembodiment, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

A method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al. Cold Spring Harbor Symp. Quant. Biol.51:263-273 (1986); European Patent 50,424; European Patent 84,796;European Patent 258,017; European Patent 237,362; European Patent201,184; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat.No. 4,683,194), using primer pairs that are capable of hybridizing tothe proximal sequences that define a polymorphism in its double-strandedform.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; and 5,616,464, all of whichare incorporated herein by reference in their entireties. However, thecompositions and methods of this invention can be used in conjunctionwith any polymorphism typing method to type polymorphisms in soybeangenomic DNA samples. These soybean genomic DNA samples used include butare not limited to soybean genomic DNA isolated directly from a soybeanplant, cloned soybean genomic DNA, or amplified soybean genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods as disclosed in U.S. Pat. No. 5,800,944 where sequence ofinterest is amplified and hybridized to probes followed by ligation todetect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al.,Bioinformatics 21:3852-3858 (2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening aplurality of polymorphisms. A single-feature polymorphism (SFP) is apolymorphism detected by a single probe in an oligonucleotide array,wherein a feature is a probe in the array. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Target nucleic acid sequence can also be detected by probe linkingmethods as disclosed in U.S. Pat. No. 5,616,464 employing at least onepair of probes having sequences homologous to adjacent portions of thetarget nucleic acid sequence and having side chains which non-covalentlybind to form a stem upon base pairing of said probes to said targetnucleic acid sequence. At least one of the side chains has aphotoactivatable group which can form a covalent cross-link with theother side chain member of the stem.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283. SBE methods are based on extensionof a nucleotide primer that is adjacent to a polymorphism to incorporatea detectable nucleotide residue upon extension of the primer. In certainembodiments, the SBE method uses three synthetic oligonucleotides. Twoof the oligonucleotides serve as PCR primers and are complementary tosequence of the locus of soybean genomic DNA which flanks a regioncontaining the polymorphism to be assayed. Following amplification ofthe region of the soybean genome containing the polymorphism, the PCRproduct is mixed with the third oligonucleotide (called an extensionprimer) which is designed to hybridize to the amplified DNA adjacent tothe polymorphism in the presence of DNA polymerase and twodifferentially labeled dideoxynucleosidetriphosphates. If thepolymorphism is present on the template, one of the labeleddideoxynucleosidetriphosphates can be added to the primer in a singlebase chain extension. The allele present is then inferred by determiningwhich of the two differential labels was added to the extension primer.Homozygous samples will result in only one of the two labeled basesbeing incorporated and thus only one of the two labels will be detected.Heterozygous samples have both alleles present, and will thus directincorporation of both labels (into different molecules of the extensionprimer) and thus both labels will be detected.

In a preferred method for detecting polymorphisms, SNPs and Indels canbe detected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescentreporter dye and a 3′quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′→3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

For the purpose of QTL mapping, the markers included should bediagnostic of origin in order for inferences to be made about subsequentpopulations. SNP markers are ideal for mapping because the likelihoodthat a particular SNP allele is derived from independent origins in theextant populations of a particular species is low, particularly ifmultiple SNPs are used in tandem to define a haplotype. As such, SNPmarkers are useful for tracking and assisting introgression of QTLs,particularly in the case of haplotypes.

The genetic linkage of additional marker molecules can be established bya gene mapping model such as, without limitation, the flanking markermodel reported by Lander et al. (Lander et al., Genetics, 121:185-199(1989)), and the interval mapping, based on maximum likelihood methodsdescribed therein, and implemented in the software package MAPMAKER/QTL(Lincoln and Lander, Mapping Genes Controlling Quantitative Traits UsingMAPMAKER/QTL, Whitehead Institute for Biomedical Research,Massachusetts, (1990)). Additional software includes Qgene, Version 2.23(1996), Department of Plant Breeding and Biometry, 266 Emerson Hall,Cornell University, Ithaca, N.Y.). Use of Qgene software is aparticularly preferred approach.

A maximum likelihood estimate (MLE) for the presence of a marker iscalculated, together with an MLE assuming no QTL effect, to avoid falsepositives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀ (MLE for the presence of a QTL/MLE given no linked QTL). TheLOD score essentially indicates how much more likely the data are tohave arisen assuming the presence of a QTL versus in its absence. TheLOD threshold value for avoiding a false positive with a givenconfidence, say 95%, depends on the number of markers and the length ofthe genome. Graphs indicating LOD thresholds are set forth in Lander etal. (1989), and further described by Arús and Moreno-González, PlantBreeding, Hayward, Bosemark, Romagosa (eds.) Chapman & Hall, London, pp.314-331 (1993).

Additional models can be used. Many modifications and alternativeapproaches to interval mapping have been reported, including the use ofnon-parametric methods (Kruglyak et al., Genetics, 139:1421-1428(1995)). Multiple regression methods or models can be also be used, inwhich the trait is regressed on a large number of markers (Jansen,Biometrics in Plant Breed, van Oijen, Jansen (eds.) Proceedings of theNinth Meeting of the Eucarpia Section Biometrics in Plant Breeding, TheNetherlands, pp. 116-124 (1994); Weber and Wricke, Advances in PlantBreeding, Blackwell, Berlin, 16 (1994)). Procedures combining intervalmapping with regression analysis, whereby the phenotype is regressedonto a single putative QTL at a given marker interval, and at the sametime onto a number of markers that serve as ‘cofactors,’ have beenreported by Jansen et al. (Jansen et al., Genetics, 136:1447-1455(1994)) and Zeng (Zeng, Genetics 136:1457-1468 (1994)). Generally, theuse of cofactors reduces the bias and sampling error of the estimatedQTL positions (Utz and Melchinger, Biometrics in Plant Breeding, vanOijen, Jansen (eds.) Proceedings of the Ninth Meeting of the EucarpiaSection Biometrics in Plant Breeding, The Netherlands, pp. 195-204(1994), thereby improving the precision and efficiency of QTL mapping(Zeng 1994). These models can be extended to multi-environmentexperiments to analyze genotype-environment interactions (Jansen et al.,Theor. Appl. Genet. 91:33-3 (1995)).

Selection of appropriate mapping populations is important to mapconstruction. The choice of an appropriate mapping population depends onthe type of marker systems employed (Tanksley et al., Molecular mappingin plant chromosomes. chromosome structure and function: Impact of newconcepts J. P. Gustafson and R. Appels (eds.). Plenum Press, New York,pp. 157-173 (1988)). Consideration must be given to the source ofparents (adapted vs. exotic) used in the mapping population. Chromosomepairing and recombination rates can be severely disturbed (suppressed)in wide crosses (adapted×exotic) and generally yield greatly reducedlinkage distances. Wide crosses will usually provide segregatingpopulations with a relatively large array of polymorphisms when comparedto progeny in a narrow cross (adapted×adapted).

Recombinant inbred lines (RIL) (genetically related lines; usually >F₅,developed from continuously selfing F₂ lines towards homozygosity) canbe used as a mapping population. Information obtained from dominantmarkers can be maximized by using RIL because all loci are homozygous ornearly so. Under conditions of tight linkage (i.e., about <10%recombination), dominant and co-dominant markers evaluated in RILpopulations provide more information per individual than either markertype in backcross populations (Reiter et al., Proc. Natl. Acad. Sci.(USA) 89:1477-1481 (1992)). However, as the distance between markersbecomes larger (i.e., loci become more independent), the information inRIL populations decreases dramatically.

Backcross populations (e.g., generated from a cross between a successfulvariety (recurrent parent) and another variety (donor parent) carrying atrait not present in the former) can be utilized as a mappingpopulation. A series of backcrosses to the recurrent parent can be madeto recover most of its desirable traits. Thus a population is createdconsisting of individuals nearly like the recurrent parent but eachindividual carries varying amounts or mosaic of genomic regions from thedonor parent. Backcross populations can be useful for mapping dominantmarkers if all loci in the recurrent parent are homozygous and the donorand recurrent parent have contrasting polymorphic marker alleles (Reiteret al. (1992)). Information obtained from backcross populations analyzedusing either codominant or dominant markers is less than that obtainedfrom F₂ populations because one, rather than two, recombinant gametesare sampled per plant.

Backcross populations, however, are more informative (at low markersaturation) when compared to RILs as the distance between linked lociincreases in RIL populations (i.e. about 0.15% recombination). Increasedrecombination can be beneficial for resolution of tight linkages, butmay be undesirable in the construction of maps with low markersaturation.

Near-isogenic lines (NIL) created by many backcrosses to produce anarray of individuals that are nearly identical in genetic compositionexcept for the trait or genomic region under interrogation can be usedas a mapping population. In mapping with NILs, only a portion of thepolymorphic loci are expected to map to a selected region.

Bulk segregant analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore et al. 1991 Proc. Natl. Acad. Sci. (U.S.A.) 88:9828-9832).In BSA, two bulked DNA samples are drawn from a segregating populationoriginating from a single cross. These bulks contain individuals thatare identical for a particular trait (resistant or susceptible toparticular disease) or genomic region but arbitrary at unlinked regions(i.e. heterozygous). Regions unlinked to the target region will notdiffer between the bulked samples of many individuals in BSA.

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar, pureline cultivar, etc). A cultivar is a race or variety of a plant speciesthat has been created or selected intentionally and maintained throughcultivation.

Selected, non-limiting approaches for breeding the plants of the presentinvention are set forth below. A breeding program can be enhanced usingmarker assisted selection (MAS) on the progeny of any cross. It isunderstood that nucleic acid markers of the present invention can beused in a MAS (breeding) program. It is further understood that anycommercial and non-commercial cultivar can be utilized in a breedingprogram. Factors such as, for example, emergence vigor, vegetativevigor, stress tolerance, disease resistance, branching, flowering, seedset, seed size, seed density, standability, and threshability etc. willgenerally dictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredaspect, a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have most attributes of the recurrentparent (e.g., cultivar) and, in addition, the desirable traittransferred from the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U.of CA, Davis, Calif., 50-98 (1960); Simmonds, Principles of cropimprovement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen,Plant breeding perspectives, Wageningen (ed), Center for AgriculturalPublishing and Documentation (1979); Fehr, In: Soybeans: Improvement,Production and Uses, 2nd Edition, Manograph., 16:249 (1987); Fehr,“Principles of variety development,” Theory and Technique, (Vol. 1) andCrop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY,360-376 (1987).

An alternative to traditional QTL mapping involves achieving higherresolution by mapping haplotypes, versus individual markers (Fan et al.2006 Genetics 172:663-686). This approach tracks blocks of DNA known ashaplotypes, as defined by polymorphic markers, which are assumed to beidentical by descent in the mapping population. This assumption resultsin a larger effective sample size, offering greater resolution of QTL.Methods for determining the statistical significance of a correlationbetween a phenotype and a genotype, in this case a haplotype, may bedetermined by any statistical test known in the art and with anyaccepted threshold of statistical significance being required. Theapplication of particular methods and thresholds of significance arewell with in the skill of the ordinary practitioner of the art.

It is further understood, that the present invention provides bacterial,viral, microbial, insect, mammalian and plant cells comprising thenucleic acid molecules of the present invention.

As used herein, a “nucleic acid molecule,” be it a naturally occurringmolecule or otherwise may be “substantially purified”, if desired,referring to a molecule separated from substantially all other moleculesnormally associated with it in its native state. More preferably asubstantially purified molecule is the predominant species present in apreparation. A substantially purified molecule may be greater than 60%free, preferably 75% free, more preferably 90% free, and most preferably95% free from the other molecules (exclusive of solvent) present in thenatural mixture. The term “substantially purified” is not intended toencompass molecules present in their native state.

The agents of the present invention will preferably be “biologicallyactive” with respect to either a structural attribute, such as thecapacity of a nucleic acid to hybridize to another nucleic acidmolecule, or the ability of a protein to be bound by an antibody (or tocompete with another molecule for such binding). Alternatively, such anattribute may be catalytic, and thus involve the capacity of the agentto mediate a chemical reaction or response.

The agents of the present invention may also be recombinant. As usedherein, the term recombinant means any agent (e.g. DNA, peptide etc.),that is, or results, however indirect, from human manipulation of anucleic acid molecule.

The agents of the present invention may be labeled with reagents thatfacilitate detection of the agent (e.g. fluorescent labels (Prober etal., Science 238:336-340 (1987); Albarella et al., European Patent144914), chemical labels (Sheldon et al., U.S. Pat. No. 4,582,789;Albarella et al., U.S. Pat. No. 4,563,417), modified bases (Miyoshi etal., European Patent 119448).

Apparatus and methods for the high-throughput, non-destructive samplingof seeds have been described which would overcome the obstacles ofstatistical samples by allowing for individual seed analysis. Forexample, U.S. patent application Ser. No. 11/213,435 (filed Aug. 26,2005) and U.S. patent application Ser. No. 11/680,611 (filed Mar. 2,2007), which are incorporated herein by reference in their entirety,disclose apparatus and systems for the automated sampling of seeds aswell as methods of sampling, testing and bulking seeds.

The disease resistant effect of the QTL can vary based on the parentalgenotype and on the environmental conditions in which the diseaseresistance effect is measured. It is within the skill of those in theart of plant breeding and without undue experimentation to use themethods described herein to select from a population of plants or from acollection of parental genotypes those that when containing a diseaselocus result in enhanced disease resistance relative to the parentgenotype.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLES Example 1. Validation of SNP Markers for Introgression of PRRResistance Loci when Used in Conjunction with Haplotype of KnownResistance Source

Phytophthora sojae is the causal agent of Phytophthora root rot (PRR)and accounts for significant soybean yield loss in the United States.Planting resistant varieties is an effective method of controlling PRR.Breeding for PRR resistant soybeans can be greatly facilitated by theuse of marker-assisted selection for PRR resistance alleles. Segregatingpopulations were generated to validate SNP markers that could be used todetect, select for, and introgress PRR resistance loci into plants

The phenotypic rating scale and definitions used herein for thefollowing Examples are included in Table 1. The percentage of plantssurviving after inoculation with P. sojae is used for classification.This rating scale provides the basis for all disease ratings anddeterminations of resistance or susceptibility in the followingExamples.

TABLE 1 Description of rating criteria used for PRR phenotyping.Phenotypic Results Rating 0-25% Dead Resistant (R) 26-49% DeadHeterozygous (H) ≥50% Dead Susceptible (S)

Segregating populations, involving crosses between a susceptiblecultivar and a series of isolines containing Rps resistance alleles,were developed for genetic mapping in the summer of 2004 (Table 2).Individual F2 plants were tissue sampled and screened with markerspolymorphic for the Rps resistant locus associated with the resistancesource. F2:3 seed of the harvested F2 plants was analyzed for the PRRreaction. Results from validation experiments demonstrating of theutility of the markers are provided in Tables 3 through 10.

TABLE 2 Screening Populations for the PRR reaction Resistant ParentResistant locus Susceptible Parent L75-3735 Rps1c MV0030 Williams 82Rps1k MV0030 L83-570 Rps3a MV0030 L89-1541 Rps3b MV0030 L92-7857 Rps3cMV0030 PI399703 Rps8 Williams

L75-3735 is a known source of Rps1c. From the L75-3735/MV0030population, a total of 322 F2 pants were tissue sampled for markerscreening. The seed harvested from each plant was used to plant andphenotype individual F3 plants from each F2-derived family for reactionto PRR. Plants exhibiting a resistant reaction were found to have adifferent haplotype from that of susceptible plants when screened withgenetic markers NS0102174, NS0118976, NS0118166, NS0099413, andNS0119335. The total number of families from the L75-3735/MV0030population with the same haplotype as the resistant parent was 41, andof these families, all 41 had a resistant reaction when phenotyped. Fromeach family, an average of 10 F2 derived F3 plants (F2 families) werephenotyped. Table 3 provides an example of the PRR reaction andhaplotype of two families from the L75-3735/MV0030 population.

TABLE 3 Validation of SNP markers used in conjunction with the haplotypeof a known Rps1c source. Total Susc. NS- NS- NS- NS- NS- Family PlantsPlants Reaction Pedigree 0102174 0118976 0118166 0099413 0119335 1 12  0R L75- GG TT TT CC TT 3735/MV0030 2 11 10 S L75- CC CC CC TT AA3735/MV0030

Williams 82 is a known source of Rps1k. From the Williams 82/MV0030population, a total of 205 F2 plants were tissue sampled for markerscreening. The seed harvested from each plant was used to plant andphenotype individual F3 plants from each F2-derived family for reactionto PRR. Plants exhibiting a resistant reaction were found to have adifferent haplotype from that of susceptible plants when screened withgenetic markers NS0118166, NS0102920, NS0114258, NS0102174, NS0119981,and NS0201536. The total number of families from the Williams 82/MV0030population with the same haplotype as the resistant parent was 24, andof these families, all 24 had a resistant reaction when phenotyped.Table 4 provides an example of the PRR reaction and haplotype of twofamilies from the Williams 82/MV0030 population.

TABLE 4 Validation of SNP markers used in conjunction with the haplotypeof a known Rps1k source. (“*” indicates a single nucleotide deletion)Total Susc. NS- NS- NS- NS- NS- NS- NS- Family Plants Plants ReactionPedigree 0118166 0102920 0114258 0118976 0102174 0119981 0201536 1 12  0R Williams82/ TT CC TT TT GG CC CGAG MV0030 2 10 10 S Williams82/ CC TTCC CC CC AA **** MV0030

L83-570 is a known source of Rps3a. From the L83-570/MV0030 population,a total of 297 F2 plants were tissue sampled for marker screening. Theseed harvested from each plant was used to plant and phenotypeindividual F3 plants from each F2-derived family for reaction to PRR.Plants exhibiting a resistant reaction were found to have a differenthaplotype than susceptible plants when screened with the genetic markersNS0101324 and NS0116265. The total number of families from theL83-570/MV0030 population with the same haplotype as the resistantparent was 64, and of these families, 58 were found to have a resistantreaction when phenotyped. Table 5 provides an example of the PRRreaction and haplotype for two families from the L83-570/MV0030population.

TABLE 5 Validation of SNP markers used in conjunction with haplotype ofa known Rps3a source. Total Susc. NS- NS- Family Plants Plants ReactionPedigree 0101324 0116265 1 11 0 R L83- AA GG 570/MV0030 2 10 9 S L83- GGTT 570/MV0030

L89-1541 is a known source of Rps3b. From the L89-1541/MV0030population, a total of 345 F2 plants were tissue sampled for markerscreening. The seed harvested from each plant was used to plant andphenotype individual F3 plants from each F2-derived family for reactionto PRR. Plants exhibiting a resistant reaction were found to have adifferent haplotype than susceptible plants when screened with thegenetic markers NS0114683, NS0102483, NS0119333, and NS0102262. Thetotal number of families from the L89-1541/MV0030 population with thesame haplotype of the resistant parent was 88, and of these families, 47were found to have a resistant reaction when phenotyped. Table 6provides an example of the PRR reaction and haplotype of two familiesfrom the L89-1541/MV0030 population.

TABLE 6 Validation of SNP markers used in conjunction with haplotype ofknown source of Rps3b. Total Susc. NS- NS- NS- NS- Family Plants PlantsReaction Pedigree 0114683 0102483 0119333 0102262 1 12  0 R L89- AA GGAA AA 1541/MV0030 2 12 12 S L89- CC AA CC GG 1541/MV0030

L92-7857 is a known source or Rps3c. From the L92-7857/MV0030population, a total of 340 F2 plants were tissue sampled for markerscreening. The seed harvested from each plant was used to plant andphenotype individual F3 plants from each F2-derived family for reactionto PRR. Plants exhibiting a resistant reaction were found to have adifferent haplotype than susceptible plants when screened with geneticmarkers NS0114683, NS0102483, NS0119333, and NS0102262. The total numberof families from the L92-7857/MV0030 population with the same haplotypeof the resistant parent was 112, and of these families, 87 were found tohave a resistant reaction when phenotyped. Table 7 provides an exampleof the PRR reaction and haplotype of two of the families from theL92-7857/MV0030 population.

TABLE 7 Validation of SNP markers used in conjunction with the haplotypeof a known source of Rps3c. Total Susc. NS- NS- NS- NS- Family PlantsPlants Reaction Pedigree 0114683 0102483 0119333 0102262 1 11  0 R L92-AA GG AA AA 7857/MV0030 2 12 11 S L92- AC AG AC AG 7857/MV0030

PI399703 is a known source of Rps8. From the PI399703/Williamspopulation, a total of 223 F2 plants were tissue samples for markerscreening. The seed harvested from each plant was used to plant andphenotype individual F3 plants from each F2-derived family for reactionto PRR. Plants exhibiting a resistant reaction were found to have adifferent haplotype than susceptible plants when screened with geneticmarkers NS0114683, NS0102483, NS011933, and NS0102262. The total numberof families from the PI399703/Williams population with the samehaplotype of the resistant parent was 39, and of these families, all 39had a resistant reaction when phenotyped. Table 8 provides an example ofthe PRR reaction and haplotype of two families from thePI399703/Williams population.

TABLE 8 Validation of SNP markers used in conjunction with the haplotypeof a known source of Rps8. Total Susc. NS- NS- NS- NS- NS- NS- FamilyPlants Plants Reaction Pedigree 0101324 0102262 0102483 0114683 01162650119333 1 6 0 R PI399703/Williams AA GG AA CC GG CC 2 4 4 SPI399703/Williams GG AA GG AA TT AA

An additional SNP marker, NS0138011, was identified for the detection ofthe Rps1c locus. Genotyping results of 100 soybean lines which wereknown to have either Rps1a, Rps1k, Rps1c, or to be susceptible to PRRwere examined. Lines with Rps1c were found to have the AA allele whenscreened with the marker NS0138011. The other 85 lines, which had eitherPRR resistance loci Rps1a, Rps1k, or were susceptible to PRR, had the CCallele as provided in Table 9. Therefore, screening with the geneticmarker NS0138011 can be used for detecting the Rps1c locus in a soybeanbreeding program.

TABLE 9 Validation of SNP marker NS0138011 in detecting Rps1c. AlleleNumber of lines Locus CC 85 Rps1a, Rps1k or susceptible AA 15 Rps1c

Table 10 summarizes validation experiments and includes the SNP markersfound to be useful in monitoring the selection or introgression of thePRR resistance locus Rps1, including Rps1c and Rps1k alleles into asoybean plant in a soybean breeding program. The haplotype of the knownresistance source is used to determine which Rps1 allele is selected forin a breeding program. Table 10 also includes SNP markers useful formonitoring the introgression of the PRR resistance loci Rps3 and Rps8into a soybean plant in a soybean breeding program. PRR resistance locusRps3 includes, Rps3a, Rps3b, and Rps3c. The haplotype of the knownresistance source is used to determine which Rps3 allele is selected forin a breeding program. SNP markers found to be useful for screening forRps1 include NS0099413, NS0102174, NS0118166, NS0102920, NS0114258,NS0118976, NS0119981, NS0119335, NS0201536, NS0138011, NS0127084,NS0129030, NS0202603, and NS0203225. SNP markers found to be useful forscreening for Rps3 and Rps8 include NS0114683, NS010324, NS0102483,NS0119333, NS0102262, and NS0116265. In a soybean breeding program,plants genotyped as homozygous or heterozygous for the resistant parentalleles, may be selected for advancement.

TABLE 10 SNP markers useful for introgression of PRR resistance lociRps1, Rps3, and Rps8 into soybean plants when used in conjunction withthe known haplotype of the resistance source. SEQ ID SEQ ID SEQ SEQ Chr.Rps SEQ SNP Forward Reverse ID ID Marker LG* Pos. Locus ID PositionAllele 1 Allele 2 Primer Primer Probe 1 Probe 2 NS0099413 N 25.0 1 1 242CC IT 17 18 49 50 NS0102174 N 25.0 1 2 563 CC GG 19 20 51 52 NS0118166 N25.0 1 3 259 CC TT 21 22 53 54 NS0102920 N 25.0 1 4 163 CC TT 23 24 5556 NS0114258 N 25.0 1 5 324 CC TT 25 26 57 58 NS0118976 N 25.0 1 6 460CC TT 27 28 59 60 NS0119981 N 28.7 1 7 516 AA CC 29 30 61 62 NS0119335 N29.5 1 8 310 AA TT 31 32 63 64 NS0202603 N 31.1 1 81 51 AA GG 85 86 9394 NS0138011 N 32.2 1 10 385 AA CC 35 36 67 68 NS0201536 N 33.0 1 935-38 CGAG **** 33 34 65 66 NS0203225 N 33.2 1 82 192 AA TT 87 88 95 96NS0129030 N 34.7 1 83 324 CC AA 89 90 97 98 NS0127084 N 41.4 1 84 858 CCTT 91 92 99 100 NS0114683 F 100.6 3.8 11 368 AA CC 37 38 69 70 NS0101324F 100.8 3.8 12 84 AA GG 39 40 71 72 NS0102483 F 101.7 3.8 13 172 AA GG41 42 73 74 NS0119333 F 101.7 3.8 14 607 AA CC 43 44 75 76 NS0102262 F103.0 3.8 15 131 AA GG 45 46 77 78 NS0116265 F 113.8 3.8 16 719 GG TT 4748 79 80 *LG = Linkage Group

Example 2: Use of SNP Markers to Select for the Rps1k Locus whichConfers Resistance to PRR Race 4

SNP markers were used with knowledge of PRR resistant germplasm to breedfor PRR resistance (Table 11). The F3 population derived from the crossof two PPR resistance sources AG3602, a source of Rps1c, and AG3505, asource of Rps1k, was screened with two SNP markers. A total of 466individuals were screened. The SNP markers NS0119335 and NS0118166 wereused to select for Rps1k based on the haplotype of AG3505, a source ofresistance for PRR race 4. Sources of Rps1 resistance include, but arenot limited to AG3602, AG3505, and DKB28-53.

TABLE 11 Haplotypes of two sources of PRR resistance. Marker 1 Marker 2Source NS0119335 NS0118166 AG3602 (Rps1c) TT CC AG3505 (Rps1k) AA TT

Example 3. Use of SNP Markers to Monitor the Introgression of PRRResistance Locus Rps1k

F3 individuals derived from a cross between two soybean lines AG3602(Rps1c) and AG3505 (Rps1k) were genotyped. The Rps1k locus providesresistance to PRR race 4. A total of 466 F3 plants are screened with twoSNP markers NS0119335 and NS0118160. Table 12 reports the results of thephenotypic validation of the genotypic screening for PRR race 4.Screening with NS0119335 was 85.7% predictive of PRR reaction. Screeningwith NS0118160 was 83.8% predictive of PRR reaction.

TABLE 12 Results from phenotypic screening following selection based onresistance source haplotype. Individuals selected Individuals Favorablebased on with Res. % Marker Haplotype haplotype Reaction ResistantOrigin NS0119335 AA 182 156 85.7 AG3602/ AG3505 NS0118160 TT 185 15583.8 AG3602/ AG3505

Example 4. Use of SNP Markers to Select for Rps3 or Rps8 which ConferResistance to PRR Race 25

F3 plants derived from the following breeding populations were genotypedwith the SNP marker NS0114683. Rps3 and Rps8 provide resistance to PRRrace 25. The breeding populations included a source of either Rps3 orRps8. Table 13 reports the results of the phenotypic validation of thegenotypic screening for PRR race 25. Screening with NS0114683 for Rps3was 96.4 to 100% predictive of the PRR reaction. Screening withNS0114683 for Rps8 was 72.4 to 80.8% predictive of the PRR reaction.

TABLE 13 SNP marker NS-0114683 was used to select plants with haplotypematching resistance source. Phenotypic screening with PRR race 25 wasthen conducted. Individuals selected Individuals Favorable based on withRes. % Haplotype haplotype Reaction Resistant Rps Origin CC 17 17 100.0Rps3 Ivory/ DKB28-53 CC 48 47 97.9 Rps3 MV0033/ CFN3303E3R CC 28 27 96.4Rps3 Ivory/ MV0036 CC 26 21 80.8 Rps8 DKB28- 53/((Darby/ OX-98317)/AG2703)) CC 29 21 72.4 Rps8 MV0039/ ((Darby/OX- 98317)/ MV0028))

Example 5. Introgression of Race Specific PRR Resistance Using SNPMarkers

In this example, L75-3735 was the source of PRR resistance locus Rps1c.Populations from a cross of L75-3735 with MV0030, a line susceptible toPRR race 3, were analyzed. Two markers, NS0099413 and NS0119335, wereused to select individuals with haplotypes matching that of theresistant parent. Individuals homozygous for the alleles present in thePRR resistant source (L75-3735) were chosen for advancement (Table 14).

TABLE 14 Use of two SNP markers to screen for resistance orsusceptibility to PRR race 3. # % Pedigree Dead Total Susc. ReactionNS0099413 NS0119335 L75-3735 (Rps1c) 0 11 0 R CC TT MV0030 9 10 90 S TTAA MV0030 5 10 50 S TT AA L75-3735/MV0030 0 10 0 R CC TT L75-3735/MV003010 10 100 S TT AA L75-3735/MV0030 5 10 50 S CT AT L75-3735/MV0030 4 1040 H CT AT

Example 6. Use of SNP Markers to Select for PRR Resistance

Further SNP markers were identified which flanked marker NS0138011. Thealleles of NS0138011 have been associated with Rps1c. The “A” allele hasbeen shown to indicate the presence of Rps1c and the “C” alleleindicates the absence of Rps1c. The combination of markers NS0202603,NS0138011, and NS0203225 demonstrate utility for distinguishing multiplealleles at the Rps1 locus including rps (susceptible), Rps1a, Rps1c, andRps1k. In a study of 239 soybean lines, the haplotypes of three markerswere effective in predicting the PRR reaction from 88 to 100% of thetime (Table 15). In a soybean breeding program, the three markers can beused to identify plants with resistance to Rps1a, Rps1c, and Rps1k, thusallowing for marker assisted selection.

TABLE 15 Ability of marker haplotypes of NS0202603, NS0138011, andNS0203225 to predict allele configuration of Rps1 for PRR resistance. #lines that show Haplotype phenotype NS0202603- of Predicted NS0138011- #lines with predicted % Rps1 allele NS0203225 haplotype allele*congruence rps GG CC AA 9  9 100 Rps1a AA CC TT 9  8 89 Rps1c AA AA AA189 186  98 Rps1k GG CC TT 32  28* 88 *3 are heterozygous based onphenotype

Example 7. Use of SNP Markers and Knowledge of Parental Genotype toIdentify Rps1c Resistant Plants

NS0129030 is useful for selecting for the Rps1c allele in populations oflate Maturity Group 3 parents that have Rps1c and are crossed tosusceptible Maturity Group 4 parents. For example, when AG3802, AG3905,AOX3903B0C, CFN3802A1X, or AG3602 are used as Rps1c donors in crosses tothe PRR-susceptible lines MV0097, MV0098, MV0099, or MV0022, all of theresistant parents have the “C” allele at NS0129030, while all of thesusceptible parents have the “A” allele. Knowledge of the resistantsource genotype is important since the association of the “C” allelewith Rps1c is not present in all other lines, as the susceptible linesMV0101, MV0102, and MV0103 also have the “C” allele at this locus.Therefore in a soybean breeding program, the marker NS0129030 can beused with knowledge of the resistant source genotype to select resistantplants.

NS0127084 is also useful in many of the same populations. For example,the Rps1c donors AG3802, AG3905, AOX3903B0C, CFN3802C1X, or AG3602 allhave the “C” allele at the NS0127084 locus, while the susceptible linesMV0097, MV0099, and MV0100 all have the “T” allele. However, MV0098 alsohas the “C” allele, but lacks Rps1c. Therefore in a soybean breedingprogram, the marker NS0127084 can be used with knowledge of theresistant source genotype to select resistant plants.

Example 8. Use of SNP Markers to Select for Rps3c

A population was provided from the cross of MV0031/BFN3205A0R, withBFN3205A0R as the heterozygous source of Rps3c which confers resistanceto Phytophthora sojae race 25. The haplotypes of the parents and that ofCFN3303E3R, a homozygous sister line of BFN3205A0R, are provided inTable 16. A total of 4,224 F2 seeds were non-destructively sampled andgenotyped. Individual seeds were selected which were homozygousfavorable for Rps3c. A total of 1018 seeds had the haplotype CCGGGG atthe marker loci NS0119333, NS0102262, NS0116265 and were selected forplanting. F2 seeds genotyped as homozygous favorable for Rps3c wereplanted in the Spring of 2006. F2:3 seed from selected plants wereplanted as progeny rows in 2006-2007. F2:4 seed bulked from selectedprogeny rows was grown in yield trials in 2007. Four lines selected forsuperior yield were evaluated for resistance to PRR race 25 to confirmpresence of Rps3c. Pathology data are provided in Table 18. Pathologytesting confirmed the marker assisted selection for resistance toPhytophthora sojae race 25. From this example, the markers provided haveshown use for marker assisted selection for PRR resistance toPhytophthora sojae race 25.

TABLE 16 Haplotypes of MV0031, BFN3205A0R, and CFN3303E3R. LineNS0119333 NS0102262 NS0116265 MV0031 AA AA GG BFN3205A0R AC AG TTCFN3303E3R CC GG GG

TABLE 17 Pathology screening for Rps3 with Phytophthora sojae race 25.Susceptible Line Replications Total Plants Plants 1 3 24 0 2 3 24 0 3 324 0 4 3 24 0

Example 9. Oligonucleotide Hybridization Probes Useful for DetectingSoybean Plants with PRR Resistance Loci

Oligonucleotides can also be used to detect or type the polymorphismsassociated with PRR resistance disclosed herein by hybridization-basedSNP detection methods. Oligonucleotides capable of hybridizing toisolated nucleic acid sequences which include the polymorphism areprovided. It is within the skill of the art to design assays withexperimentally determined stringency to discriminate between the allelicstates of the polymorphisms presented herein. Exemplary assays includeSouthern blots, Northern blots, microarrays, in situ hybridization, andother methods of polymorphism detection based on hybridization.Exemplary oligonucleotides for use in hybridization-based SNP detectionare provided in Table 18. These oligonucleotides can be detectablylabeled with radioactive labels, fluorophores, or other chemiluminescentmeans to facilitate detection of hybridization to samples of genomic oramplified nucleic acids derived from one or more soybean plants usingmethods known in the art.

TABLE 18 Oligonucleotide Hybridization Probes* Marker SNP SEQ ID MarkerSEQ ID Position Hybridization Probe Probe NS0119335 8 310TCTCAGAGTGGGTAGA 101 NS0119335 8 310 TCTCAGTGTGGGTAGA 102 NS0138011 10385 GAATGAAAAATCTACT 103 NS0138011 10 385 GAATGACAAATCTACT 104 NS011933314 607 TAAGAACCCTCTCCAA 105 NS0119333 14 607 TAAGAAACCTCTCCAA 106NS0102262 15 131 AAGCCTGACAATTGAT 107 NS0102262 15 131 AAGCCTAACAATTGAT108 *16 mer spanning SNP

Example 10. Oligonucleotide Probes Useful for Detecting Soybean Plantswith PRR Resistance Loci by Single Base Extension Methods

Oligonucleotides can also be used to detect or type the polymorphismsassociated with PRR resistance disclosed herein by single base extension(SBE)-based SNP detection methods. Exemplary oligonucleotides for use inSBE-based SNP detection are provided in Table 19. SBE methods are basedon extension of a nucleotide primer that is hybridized to sequencesadjacent to a polymorphism to incorporate a detectable nucleotideresidue upon extension of the primer. It is also anticipated that theSBE method can use three synthetic oligonucleotides. Two of theoligonucleotides serve as PCR primers and are complementary to thesequence of the locus which flanks a region containing the polymorphismto be assayed. Exemplary PCR primers that can be used to typepolymorphisms disclosed in this invention are provided in Table 10 inthe columns labeled “Forward Primer SEQ ID” and “Reverse Primer SEQ ID”.Following amplification of the region containing the polymorphism, thePCR product is hybridized with an extension primer which anneals to theamplified DNA adjacent to the polymorphism. DNA polymerase and twodifferentially labeled dideoxynucleoside triphosphates are thenprovided. If the polymorphism is present on the template, one of thelabeled dideoxynucleoside triphosphates can be added to the primer in asingle base chain extension. The allele present is then inferred bydetermining which of the two differential labels was added to theextension primer. Homozygous samples will result in only one of the twolabeled bases being incorporated and thus only one of the two labelswill be detected. Heterozygous samples have both alleles present, andwill thus direct incorporation of both labels (into different moleculesof the extension primer) and thus both labels will be detected.

TABLE 19 Probes (extension primers) for Single BaseExtension (SBE) assays. Marker SNP SEQ ID Marker SEQ ID PositionSBE Probe Probe NS0119335 8 310 AGACTCTCTCTCTCAGA 109 NS0119335 8 310ATTGGATTTCTACCCAC 110 NS0138011 10 385 AAATTCCTGTGAATGAA 111 NS013801110 385 TTATTCAAAGTAGATTT 112 NS0119333 14 607 TTTTTTAAATTAAGAAC 113N50119333 14 607 TGAAAGTGTTGGAGAGG 114 NS0102262 15 131AAGTACTCCCAAGCCTG 115 NS0102262 15 131 ACAAGACAATCAATTGT 116

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1-25. (canceled)
 26. A method of introgressing at least one PRRresistance QTL into a soybean plant comprising a. crossing a PRRresistant soybean plant with a second soybean plant to form apopulation; b. screening said population with at least one nucleic acidmarker selected from the group consisting of SEQ ID NO: 1-16, and SEQ IDNO: 80-84; c. selecting from said population one or more soybean plantscomprising at least one genotype corresponding to the PRR resistantsoybean plant.
 27. The method according to claim 26, wherein at least51% or at least 75% of the selected soybean plants exhibit a resistantreaction to PRR.
 28. (canceled)
 29. The method of claim 26, furthercomprising the step (d) of assaying said selected soybean plant forresistance to a PRR inducing pathogen.
 30. The method of claim 26,wherein said genotype is determined by an assay which is selected fromthe group consisting of single base extension (SBE), allele-specificprimer extension sequencing (ASPE), DNA sequencing, RNA sequencing,microarray-based analyses, universal PCR, allele specific extension,hybridization, mass spectrometry, ligation, extension-ligation, and FlapEndonuclease-mediated assays.
 31. The method of claim 26, furthercomprising the step of crossing the soybean plant selected in step (c)to another soybean plant.
 32. The method of claim 26, further comprisingthe step of obtaining seed from the soybean plant selected in step (c).33. An elite soybean plant produced by the method of claim
 26. 34-38.(canceled)
 39. A substantially purified nucleic acid molecule for thedetection of loci related to PRR resistance comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1 through SEQID NO: 116 and complements thereof.
 40. (canceled)
 41. A method ofintrogressing an allele into a soybean plant comprising: a. Providing apopulation of soybean plants, b. genotyping at least one soybean plantin the population with respect to a soybean genomic nucleic acid markerselected from the group SEQ ID NO: 1 to SEQ ID NO: 16 and SEQ ID NO: 80to SEQ ID NO: 84 and c. selecting from said population one or moresoybean plants comprising a haplotype associated with PRR resistance,wherein said PRR resistance haplotype is selected the group consistingof 1, 2, or 3 PRR resistant loci where one or more haplotypes at one ormore loci are selected from the group consisting of Rps1, Rps3, andRps8, and the one or more haplotypes are selected based on the haplotypeof the PRR resistant soybean plant.
 42. The method of claim 41 in whichproviding a population comprises crossing a PRR resistant soybean plantwith a second soybean plant to form a population.
 43. The methodaccording to claim 41, wherein said selected one or more soybean plantsexhibit increased grain yield in the presence of PRR as compared tosoybean plants lacking PRR resistance loci.
 44. The method according toclaim 43, wherein said selected one or more soybean plants exhibit anincreased grain yield of at least 0.5 Bu/A, at least 1.0 Bu/A or atleast 1.5 Bu/A in the presence of PRR as compared to soybean plantslacking PRR resistance loci. 45-46. (canceled)